Biological systems have developed antioxidant systems to combat the effects of radicals and other pro-oxidative species. An antioxidant is any substance that significantly delays or prevents oxidation of an oxidizable substrate. Certain enzymes are antioxidants such as superoxide dismutase and catalase, which are coded for by many organisms. Substances such as vitamin C and plant phenols are antioxidants introduced through the diet into biological systems. It has been proposed that naturally occurring levels of these substances are not adequately produced in the body or ingested in the normal diet. The normal diet often does not provide enough antioxidants because it is deficient in fruits and vegetables. Further, fruits and vegetables that are in the diet are likely depleted of their antioxidants due to modern-day processing. The normal diet could be improved, but taking into account today's lifestyles and poor composition of Western foods many choose to supplement their normal diet with antioxidants.
While a tremendous amount of research has gone into use of antioxidants as nutritional supplements, supplementation of the modern diet has not been consistently successful (European Research on Functional Effects of Dietary Antioxidants, Sep. 25-28, 2002, Cambridge, UK). Problems with accurately and consistently measuring antioxidant capacity under various conditions and of measuring compositions having varying amounts of lipophilic versus hydrophilic ingredients have not been solved.
Several laboratory methods have been developed to determine the ability of a substance to quench a free radical or to determine its antioxidant capacity. For a review, see Halliwell B., and Gutteridge J. M. C., Free Radicals in Biology and Medicine. 3rd Edition. New York: Oxford University Press, 2000. These assays are the TEAC, 19F-NMR, TRAP, modified TRAP, FRAP, the Glazer fluorescence-based method, the phosphomolybdenum complex method, and the ORAC, all of which are proposed to measure some aspect of a substance's ability to quench free radical species.
Prior ORAC assays for antioxidant capacity rely on the generation of a radical that oxidizes a substrate. In the presence of an antioxidant, the oxidation of the substrate is slowed. The protective effect of an antioxidant is measured by determining the area under a decay curve (AUC) of the test sample as compared to a blank. A method called oxygen radical absorbance capacity (ORAC (β-PE)) measures antioxidant activity against peroxyl radical induced by 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) at 37° C. β-phycoerythrin (β-PE) was used in these assays as a fluorescent probe where loss of fluorescence of β-PE is an indication of the extent of damage from its reaction with a peroxyl radical.
Several drawbacks of using β-PE as a probe led Ou et al. (J Agric Food Chem. 2001; 49(10):4619-4626) to develop an assay using fluorescein as the fluorescent probe (ORAC (fl)). However, the fluorescein system does not measure antioxidant capacity of lipophilic samples since the assay is performed in aqueous solution. A modification of the ORAC (fl) method was described by Huang et al. (J Agric Food Chem. 2002; 50(7):1815-1821) for lipophilic antioxidants using randomly methylated beta-cyclodextrin as a solubility enhancer. The reaction mechanism was determined to be that of hydrogen atom transfer. The ORAC (fl) method using beta-cyclodextrin does not measure antioxidant activity of carotenoids and polyunsaturated fatty acids since carotenoids and fatty acids are not chain-breaking antioxidants (Huang et al. ibid). These antioxidants may act as singlet oxygen scavengers and may follow a different reaction mechanism. The ORAC (fl) method has been accepted by the industry as the gold standard for measuring antioxidant capacity.
U.S. published patent application 2002/0182736 to Aldini et al. describes a method that is alleged to determine the total antioxidant activity of a sample in both lipid and aqueous compartments. The sample is incubated with a lipophilic radical generator and an oxidizable lipophilic indicator in a lipid compartment of the sample. Oxidation of the oxidizable lipophilic indicator provides a measure of the antioxidant activity of the lipid compartment of the sample. The sample is also incubated with a hydrophilic radical generator and an oxidizable hydrophilic indicator in an aqueous compartment of the sample. Oxidation of the oxidizable hydrophilic indicator provides a measure of the antioxidant activity of the aqueous compartment of the sample. HPLC and fluorescence assays were used to determine these separate values.
The present inventors observed that assays using the ORAC (fl) provided data that showed an increase in fluorescence after addition of the radical initiator. The ORAC (fl) relies on the assumption that fluorescein is the only fluorescent component being measured. In fact, any molecule having absorbance, fluorescence or phosphorescence at the wavelength measured in the ORAC (fl) contributes to the values obtained from this assay. Further, such measured absorbance, fluorescence, or phosphorescence may not be related to antioxidant capacity as demonstrated by initial rise in fluorescence values. Therefore, any compound in a complex sample that fluoresces at the same wavelength as fluorescein will also be measured causing a skewed result.
Oxygen uptake has been previously used as a measure of antioxidant activity. The discovery that aqueous dispersions of oxidizable organic compounds can be readily and reproducibly initiated at a constant rate, Ri, by peroxidation using the water-soluble azo compound 2,2′-azo-bis-(2-amidipropane hydrochloride), ABAP, is the basis of the Total Radical-Trapping Antioxidant Parameter, TRAP, method developed by Wayner et al. (FEBS Lett. 1985; 187(1):33-37). Upon addition of the ABAP to plasma, the length of time that oxygen uptake by peroxidizable plasma is inhibited is measured with an oxygen probe, and this value is referred to as the TRAP. Trolox (a synthetic antioxidant used as a standard) is used in this method to induce a second reaction period after the natural antioxidants have been depleted. This second induction period is used to calculate an Ri value, which is used to calculate the TRAP value. The TRAP value is reported as the number of moles of peroxyl radicals trapped per liter of fluid.
The duration of time is the only parameter measured in the TRAP method and is a limiting factor in its analysis. The time taken to prevent maximum oxygen uptake cannot be measured easily and precisely, and the total radical trapping capability per mole of some antioxidants is dependent on their initial concentration. This analysis leaves out the extent of inhibition.
A modified TRAP assay was later published by Ghiselli et al. (Free Radic. Biol. Med. 1995; 18(1):29-36), which corrects for interferences from plasma proteins or sample dilution. This modified method is based on the ability of plasma to protect β-PE from peroxyl radical attack produced by ABAP. Protection is accomplished by precipitating the protein out of plasma with ammonium sulfate and ultracentrifugation. The modified TRAP assay is performed by adding the reagents to quartz fluorometer cells and, after a 37° C. incubation for 5 minutes, ABAP is added. Fluorescence is monitored at 495 nm every 5 minutes maintaining the same temperature throughout the experiment.
This modified TRAP method produces a linear decrease in fluorescence due to the thermal decomposition of ABAP. A period of total protection is indicated by a lag phase upon the addition of any antioxidant compound. It is assumed that the total plasma antioxidant capacity is directly related to the length of the lag phase. The TRAP is quantified by comparing the lag phase produced by the antioxidant compound to the lag phase produced by a Trolox solution of known concentration. The modified TRAP method does not measure the ability of plasma to break the lipid peroxidation chain triggered by ABAP, and it is not fully defined whether and to what extent lipid-soluble antioxidants are involved in TRAP.
The present invention addresses these problems in the art and provides an assay that measures oxygen uptake of a complete system, allowing for selection of optimum ratios of ingredients for an antioxidant composition. An optimized synergistic antioxidant composition is also provided.